Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that have been known to case sporadic outbreaks in Africa and Southeast Asia. Although ZIKV disease is primarily spread to people through the bite of an infected mosquito of the Aedes specie, recent evidence indicates that the disease can also be transmitted by sexual contact. Recently, ZIKV has been linked to Guillain-Barre syndrome and to microcephaly in infants of infected mothers, a condition where infants are born with abnormally small heads. The explosive recent pandemic of ZIKV infections throughout South and Central America, the South Pacific and the Caribbean, and the potential threatening to the United States represents the most recent unexpected arrival of an arthropod-borne viral disease in the Western Hemisphere over the past 20 years. Moreover, increase in temperature will impact the spread of the Aedes mosquito and also ZIKV disease. Currently, there are no FDA- licensed vaccines or antivirals to combat ZIKV infection in humans. The significance of ZIKV in human health and the lack of prophylactic (vaccines) and therapeutic (antivirals) methods to combat ZIKV infection, highlight the importance of developing safe and effective countermeasures to combat ZIKV- induced disease in humans. The central goal of this application is to test the novel hypothesis that a single-cycle infectious (SCI) virus based-approach can be used to develop a safe, immunogenic and protective live-attenuated vaccine (LAV) to combat disease caused by ZIKV infection in both, fetuses and pregnant women. The bases for our hypothesis are: 1) existing evidence that this approach can be used to effectively generate different SCI RNA viruses, including flaviviruses; 2) immunization of validated animal models with SCI RNA viruses, including flaviviruses, has been shown to be safe and able to provide, upon a single immunization dose, complete protection against a subsequent lethal challenge with wild-type (WT) forms of the viruses; 3) SCI and WT viruses have the same protein sequence and hence identical immunogenic properties; 4) SCI RNA viruses contain a deletion of the viral envelope glycoprotein, thus making reversion to a WT virulent virus impossible; and 5) generation of SCI RNA viruses can be rapidly achieved combining the use of reverse genetic approaches with constitutively expressing viral glycoprotein-expressing cell lines. To test our hypothesis, we propose to develop reverse genetic approaches for ZIKV (Aim 1) that will be used to generate SCIrZIKVs (Aim 2). These SCIrZIKVs will be used to develop a safe, immunogenic and protective LAV (Aim 3) to combat ZIKV disease in fetuses and pregnant mice (Aim 4). The knowledge we have accumulated in both, the use of RNA virus reverse genetic techniques (including flaviviruses) and the implementation of SCI viruses for the generation of LAVs demonstrate that we have the expertise and knowledge needed for the successful implementation of this approach for ZIKV.